RNA was extracted by the CTAB-based method as described [64 (link)]. cDNAs were produced using the MMLV Reverse transcription PCR Kit with oligo(dT)15 (RT-PCR, Evrogen, Moscow, Russia) as described [65 (link)].
The mRNA transcript levels of the transgenes were determined by real-time quantitative PCR (qRT-PCR)—the 2−ΔΔCT method [66 (link)] with two internal controls, including AtGAPDH (NM_111283.4) and AtEF (XM_002864638) [65 (link)]. The primers designed for qRT-PCRs are shown inTables S1 and S2 .
qRT-PCR reactions were performed in volumes of 20 µL using the real-time PCR kit (Evrogen), as described [65 (link)], containing 1x Taq buffer, 2.5 mM MgCl2, 0.2 mM of each dNTP, 0.2 µM of each oligonucleotide primer, 1x SybrGreen I Real-time PCR dye, 1 µL cDNAs, and 1 unit of Taq DNA polymerase (Evrogen). Analysis was performed in DTprime 4M1 Thermal Cycler (DNA-technology, Moscow, Russia) programmed for an initial denaturation step of 2 min at 95 °C followed by 50 cycles of 10 s at 95 °C and 25 s at 62 °C.
The mRNA transcript levels of the transgenes were determined by real-time quantitative PCR (qRT-PCR)—the 2−ΔΔCT method [66 (link)] with two internal controls, including AtGAPDH (NM_111283.4) and AtEF (XM_002864638) [65 (link)]. The primers designed for qRT-PCRs are shown in
qRT-PCR reactions were performed in volumes of 20 µL using the real-time PCR kit (Evrogen), as described [65 (link)], containing 1x Taq buffer, 2.5 mM MgCl2, 0.2 mM of each dNTP, 0.2 µM of each oligonucleotide primer, 1x SybrGreen I Real-time PCR dye, 1 µL cDNAs, and 1 unit of Taq DNA polymerase (Evrogen). Analysis was performed in DTprime 4M1 Thermal Cycler (DNA-technology, Moscow, Russia) programmed for an initial denaturation step of 2 min at 95 °C followed by 50 cycles of 10 s at 95 °C and 25 s at 62 °C.
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